The slow pace of antibiotic discovery is being outmatched by rapid acquisition of resistance by our pathogens. The need is especially acute in the case of Mycobacterium tuberculosis (TB), where strains of XDR-TB resistant to the majority of standard therapeutics are rapidly spreading. In a previous RO1 screen development project, we collaborated with Professor Kim Lewis (Northeastern University) to establish a drug discovery platform for identifying compounds specifically acting against TB. The platform is based on two innovations - the use of uncultured bacteria as an untapped source of secondary metabolites; and a species-specific screen. Uncultured bacteria represent 99% of all species, and we access them by cultivation in their natural environment, the soil, in a specialized diffusion chamber. Our findings show that this is an excellent source of novel antimicrobials. However, even with this previously inaccessible resource most of the effort is wasted on rediscovering known compounds. We reasoned that the background can be eliminated if discovery is focused on a species-specific compound. Natural compounds specifically acting against TB are essentially unknown, so a screen for specific anti-TB compounds will produce hits that will have a high probability of being novel substances. A TB-specific compound will likely not hit a target present in humans, and will not harm the human microbiome. We performed a pilot screen against TB and a counter screen against Staphylococcus aureus. The screen produced a hit rate of 0.34% for TB-specific extracts. One of the extracts from a rare Actinomycete contained a novel antimicrobial with excellent activity against TB, high specificity for mycobacteria, and no cytotoxicity. This compound, which we named lassomycin, killed both growing and dormant cells of TB better than the best current killing agent, rifampicin. Whole genome sequencing of lassomycin-resistant mutants showed that it targets the C1 subunit of the essential mycobacterial ClpP1P2C1. Lassomycin inhibits this protease and activates the ATPase of ClpC1. This dual action is responsible for the excellent killing. Lassomycin serves as proof of principle for this platform. In the proposed project, the PI, Losee Ling, VP of Biology at NovoBiotic, will continue collaborating with Dr. Kim Lewis to perform a large scale screen of extracts from uncultured bacteria for TB-specific compounds. The compounds will be isolated, structurally characterized, and tested against a panel of gut symbionts to establish novelty and specificity. The principal mode of action will be identified by whole genome sequencing of resistant mutants. The result of the project will be eight validated leads, providing a solid basis for a subsequent Phase II effort aimed at drug development.